All references cited herein are expressly incorporated herein by reference in their entirety.
Site-specific selective protein modification procedures have been useful for oriented protein immobilization, for studies of naturally occurring post-translational modifications, for creating antibody-drug conjugates, for the introduction of fluorophores and other small molecules on to proteins, for examining protein structure, folding, dynamics, and protein-protein interactions, and for the preparation of protein-polymer conjugates. One approach for protein labeling is to incorporate biorthogonal functionalities into proteins at specific sites via enzymatic reactions. The incorporated sites then support chemoselective reactions, since reactions may be defined that are inert to normal biological materials, and occur selectively when the biorthogonal component is present. Known enzymes for site-specific ligation include formylglycine generating enzyme, sialyltransferases, phosphopantetheinyltransferases, O-GlcNAc post-translational modification, sortagging, transglutaminase, farnesyltransferase, biotin ligase, lipoic acid ligase, and N-myristoyltransferase.
Proteins, comprised of varying numbers of 20 distinct amino acid residues, arranged in a specific sequence, are the primary mediators of biological processes in all organisms, from single cell bacteria to humans. Techniques to manipulate the function of proteins can therefore find important applications in fundamental science as well as medicine and engineering. For example, the capacity to attach therapeutic chemical matter to specific amino acid residues in an antibody can pave the way for targeted therapeutics (i.e. antibody-drug conjugates). In addition, techniques to attach fluorophores or other optical probes to specific amino acid residues of an enzyme can prove useful for investigating the protein's spatial and temporal function in a specific biological process.
Joining together chemical matter with a protein (i.e. conjugation) requires at a minimum two reactive functional groups, such as a nucleophile and an electrophile, that, when combined in solution, chemically unite. Because proteins are metastable, suitable conjugation chemistry must involve functional groups that react together selectively without appreciable side-reactions; tolerate the presence of water, salts, and buffers; proceed at reasonable rates at ambient temperature; and progress to near completion so as to minimize post-reaction workup of the conjugated protein.
There are several proteins with conjugation activity that have been developed commercially. Prominent examples: Halotag (Promega) www.promega.com/products/pm/halotag-technology/halotag-technology/ SNaP tag (New England Biolabs) www.neb.com/applications/protein-analysis-and-tools/proteinlabeling/protein-labeling-snap-clip Biotin ligase (Avidity) www.avidity.com/technologies/vitrobiotinylation-avitag-enzyme Sfp phosphopantetheinyltransferase (New England Biolabs) www.neb.com/products/p9302-sfp-synthase.
Of specific interest here is the conjugation of proteins to nucleic acids. Protein-DNA conjugates have been sought for fundamental and applied studies. In his 2010 review Niemeyer, Christof M. “Semisynthetic DNA-protein conjugates for biosensing and nanofabrication.” Angewandte Chemie International Edition 49, no. 7 (2010): 1200-1216 (www.ncbi.nlm.nih.gov/pubmed/20091721), Niemeyer identified a number of emerging areas, including bioanalytics (i.e immunoPCR); DNA directed immobilization of proteins (biochips); nanofabrication of protein assemblies (DNA arrays); and synthesis of medicinal nanoparticles bearing therapeutic proteins and peptides.
Current methods to conjugate proteins with nucleic acids depend on “spontaneous” chemical conjugation chemistry, such as disulfide bond formation, as opposed to conjugation catalyzed by a biomolecule. No enzyme has been fully described that can directly conjugate proteins with nucleic acids. Rather, methods in current use require installing reactive functional groups on the protein and separately, on the nucleic acid (Neimeyer, www.ncbi.nlm.nih.gov/pubmed/20091721). The chemically modified protein and nucleic acid are combined in a single tube and allowed to react. Often, these bimolecular reactions proceed slowly, requiring 24 h or more, and generate modest yields, see for example (Barbuto, Scott, Juliana Idoyaga, Miguel Vila-Perelló, Maria P. Longhi, Gaëlle Breton, Ralph M. Steinman, and Tom W. Muir. “Induction of innate and adaptive immunity by delivery of poly dA:dT to dendritic cells.” Nature chemical biology 9, no. 4 (2013): 250-256, www.ncbi.nlm.nih.gov/pubmed/23416331). More recently, strategies have emerged that make use of activated esters as well as click chemistry, and appear to increase the speed conjugation; however they suffer from a lack of specificity with respect to the site (i.e. amino acid residue) of protein-nucleic acid conjugation (www.ncbi.nlm.nih.gov/pubmed/26947912).
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